Devices and methods for controlling the optical power in a microscope are known in the field. In the devices of the type described, fast acousto-optical or electro-optical elements are generally used to adjust the optical power of an illuminating light beam in a nearly infinitely variable and spectrally selective manner. The optical elements used here are primarily AOTF (acousto-optical tunable filter) crystals which allow the optical power of a laser used as a light source in a microscope to be controlled for each wavelength. To this end, generally, an RF frequency corresponding to the desired laser wavelength and a corresponding amplitude of the RF wave are applied to the AOTF crystal via a control unit.
The operating principle of an AOTF crystal is based on the fact that, for example, the RF frequency applied to the crystal acts as an optical grating for a laser beam incident perpendicular to the RF frequency, allowing the incident laser light to be diffracted into the first order maximum in a nearly completely collinear manner, and thus to be provided as an illuminating light beam. By linear superposition of different RF frequencies, laser light of different wavelengths can be collinearly diffracted and picked off with different intensities at the AOTF.
A crucial parameter of acousto-optical elements is the sound velocity, i.e., the velocity at which the RF wave applied to the crystal propagates in the crystal. A change in the sound velocity results in a change in the diffraction efficiency of the crystal, i.e., the laser wavelength diffracted with maximum intensity is shifted in frequency.
A device of the type described is known, in particular, from German Patent DE 198 27 140 C2. The laser scanning microscope described therein has also an AOTF crystal provided in the input laser beam path for spectrally selective adjustment of the optical power of the illuminating light beam. Since the sound velocity in the AOTF crystal is temperature-dependent, a temperature sensor is provided in the vicinity of the crystal, the temperature sensor registering the temperature as a measuring signal. To maintain the optical illumination power constant, it is proposed there as a first measure to maintain the AOTF crystal at a constant temperature using heating control means. As an alternative measure, it is proposed to control the AOTF frequency via a control unit as a function of the detected temperature to thereby correct a change in the optical power of the illuminating light beam resulting from the temperature change.
To carry put such a correction, calibration curves are needed from which can be derived the relationship between a change in the temperature of the crystal and a resulting change in the diffraction efficiency or a shift in frequency of the optimally diffracted laser wavelength.
This method has the problem that, on the one hand, it only relies on the correctness of the underlying calibration curve and, on the other hand, that measuring errors in the calibration curves will propagate. Moreover, its use in practice is extremely inflexible because a new calibration curve must first be generated for each illumination wavelength.
A further problem is that the actual crystal temperature can only be measured with a delay in time. Due to the dimensions of the crystal, the time constant is typically several minutes. However, due to the absorption of the RF power in the crystal, the temperature can also change on a shorter time scale so that inaccuracies may creep in this manner as well.